Disclosed is a gene which encodes an alanyl-glutamine dipeptide biosynthetic enzyme and an application thereof, the nucleotide sequence of said gene being shown in SEQ ID NO. 1. Further disclosed is the amino acid sequence encoded by the gene, and provided are a recombinant vector containing the gene, recombinant E. coli, recombinant S. cerevisiae, and self-flocculating recombinant S. cerevisiae. Further disclosed is a method for carrying out bioconversion to synthesize alanyl-glutamine dipeptide by using said recombinant microorganisms.